The overall objective of this proposal is to apply an anti-idiotype (Id) based vaccine approach for the treatment of HER2/neu positive human cancer. Amplification and/or overexpression of HER2/neu occurs in multiple human malignancies and is associated with a poor prognosis. A humanized anti-HER2/neu mAb, 4D5 (Herceptin) is therapeutic when administered for a subset of patients with metastatic breast cancer, especially in combination with chemotherapy. These exciting results provide a basis for further development of new antibody-based cancer therapy strategies. One such strategy is active specific immunotherapy in which the host immune system is targeted to HER2/neu expressing tumor cells. We propose to develop "internal image" anti-Id vaccines that will resemble the three- dimensional shapes of HER2/neu. Immunization with such an anti-Id vaccine will break immune tolerance and induce sustained high titer HER2/neu specific antibodies in patients in a genetically unrestricted fashion. We will target the clinically proven, protective epitopes of HER2/neu as defined by Herceptin and also by mAb 520C9 which recognizes a different epitope. Murine mAb 4D5 (parental antibody for Herceptin) and 520C9 will be used separately as the immunizing antibody or Abi to which anti-Ids or Ab2 will be generated. Ab2 will be used to induce anti-HER2/neu antibodies (Abi') in mice and rabbits. The cross-reactivities of the induced anti-anti-Id (Ab3) antibodies with the native HER-2 receptor will be determined by flow cytometry, immunohistochemistry, and immunoprecipitation with the use of corresponding Abi as positive control. The anti-tumor activity of Ab3 (Abi') will be assessed both in vitro and invivo. We will determine the Ab3 mediated growth inhibitory effects on HER2/neu positive cancer cells in tissue culture and in xenografted nude mice. The cellular and molecular mechanisms of antibody-induced growth inhibition will be investigated. The therapeutic efficacy of the potential anti-Id vaccines will then be tested for tumor protection as well as therapy of established tumors in an immunocompetent murine model of carcinoma cells transduced with the HER2/neu gene. Immunotherapeutic potential of the selected anti-Id vaccines will also be evaluated in HER2/neu transgenic mice. The criteria for selection of optimal anti-Id vaccines will be based on the ability to invoke anti-tumor activities in vitro and invivo. This study will be a prelude to clinical trials for HER2/neu positive cancer patients with a single or combined anti-Id vaccine.